A common neuronal ensemble in nucleus accumbens regulates pain-like behaviour and sleep

A comorbidity of chronic pain is sleep disturbance. Here, we identify a dual-functional ensemble that regulates both pain-like behaviour induced by chronic constrictive injury or complete Freund’s adjuvant, and sleep wakefulness, in the nucleus accumbens (NAc) in mice. Specifically, a select population of NAc neurons exhibits increased activity either upon nociceptive stimulation or during wakefulness. Experimental activation of the ensemble neurons exacerbates pain-like (nociceptive) responses and reduces NREM sleep, while inactivation of these neurons produces the opposite effects. Furthermore, NAc ensemble primarily consists of D1 neurons and projects divergently to the ventral tegmental area (VTA) and preoptic area (POA). Silencing an ensemble innervating VTA neurons selectively increases nociceptive responses without affecting sleep, whereas inhibiting ensemble-innervating POA neurons decreases NREM sleep without affecting nociception. These results suggest a common NAc ensemble that encodes chronic pain and controls sleep, and achieves the modality specificity through its divergent downstream circuit targets.

pain-labeled D1 neurons significantly different from other NAc D1 neurons because the previous study showed that NAc D1-VTA projection promotes wakefulness (Luo, 2018). If yes, it is necessary to show the difference. In addition, whether optogenetic activation of NAc-VTA projection increases the activity of VTA dopaminergic and glutamatergic neurons? It's better to check the activity of these neurons after optogenetic activation to further explain the downstream circuit mechanisms. 2. Figure 1F & 2D, a significant 10 Hz signal in EEG heatmap. Is it from opto-stimulation? However Figure  1L did not show it. Noise elimination is needed to increase the reliability of results. 3. In Figure S3, the firing rate of NAc D1 neurons was not altered in sleep-wake transitions in naïve mice, do the author think these neurons are not involved in the regulation of wakefulness? How to understand the increased calcium activity of NAc neurons in wakefulness from the previous study? 4. Figure 6H, why these VTA and POA-projecting cells are normalized to total VTA and POA cells, but not NAc cells? 5. Inhibition experiments in Figure 7 are indirect evidence. Whether the stimulation of NAc to POA and VTA projections will get similar results?
Reviewer #3 (Remarks to the Author): This is an elegant study that significantly advances our knowledge about the interplay between sleep and pain. I will summarize the main data to let the authors know what I believe they should consider before publication.
Sun and cols. used c-fos-tTA transgenic mice to express several AAV-carried effect genes under the c-fos promoter, so that the transgenes were preferentially expressed in neurons with high expression of c-Fos in the target region. The authors focused on the Nucleus accumbens (NAc) an interface region in the control of movement, pain, and sleep-wake cycle. The expression of the controlled transgenes was released (by terminating doxycycline supply) between day 0 and 21 after CCI (chronic constriction of the sciatic nerve) or CFA (Complete Freund's adjuvant) injections. So, NAc neurons activated in these chronic pain models were targeted.
These neurons showed naturally increased activity during nociceptive tests and wakefulness (with decreased activity during non-REM sleep (NREM sleep)) (Fig 1). The optogenetic stimulation of these neurons further decreases mechanical paw withdrawal threshold (PWT) and thermal latency (PWL); and increases wakefulness (decreasing NREM sleep) in CCI or CFA animals (Fig 2 and 3). Complementary, their optogenetic inactivation induces the opposite effects (Fig 4). The majority (80%) of CCI target neurons express dopamine D1 receptors and optogenetic stimulation of these D1 neurons (using D1-Cre mice) decreased both the PWT and PWL and increased the percentage of wakefulness (decreasing sleep); while their optogenetic inactivation increases the PWT and PWL and increases sleep (Fig 5). Anterograde tracing of NAc target neurons identified several downstream brain regions that are known to regulate sleep/wakefulness and/or nociception and a double-labeling assay suggest that these neurons are GABAergic principal medium spiny neurons. The authors focused in VTA (ventral tegmental area) and POA (preoptic area) and used a retrogradely label strategy to demonstrate that a portion (30%) of NAc target neurons divergently projected to both POA and VTA (fig 06). Finally, the authors used a dual-viral system to optogenetically inactivate the VTA or POA neurons innervated by the NAc targeted neurons. Inactivation of POA neurons decreases sleep (with no effect on nociceptive tests), while inactivation of VTA neurons decreases PWT and PWL (with no effect on sleep) (fig 07). Based on this broad set of data, the authors concluded that these NAc-targeted neurons form "… a common NAc ensemble that encodes chronic pain and controls sleep" The bidirectional relationship between negative changes in sleep and pain has been the focus of several studies in the last two decades and the NAc is emerging as a key candidate to mediate the effect of decreased sleep on pain processing. However, the underlying mechanisms are poorly understood, and this is the first study to identify a neural population in the NAc that is apparently involved in the control of pain and sleep/wake cycle. These data will impact the literature on the field and are at the edge of knowledge. I have one main concern about the experiments and some suggestions to improve the manuscript reading and data presentation Major How are the authors sure that the NAc targeted neurons does not simply control the movement of paw withdrawal after mechanical or thermal stimulation in CCI or CFA animals? This is important not only in view of the role of NAc (especially D1 neurons) in initiating movement, but also because paw withdrawal was consistently evoked throughout the experiments with no apparent need, since these behavioral data (days 3 to 21) were not shown. Indeed, it is unfortunate that the authors did not perform a single experiment to assess motor functon(another than PW) upon targeted neurons manipulation. I believe that the absence of PW changes in sham animals does not solve the problem because they are not under neuropathic (CCI) or inflammatory (CFA) sensitization. Some kind of motor function monitoring would also be welcome during optogenetic stimulation that induces the transition from sleep to wakefulness. This reviewer would like to see at least one nociceptive experiment performed without motor bias. Conditioned place preference (CPP) under target neurons inhibition would be a great alternative and it is feasible in CCI model. The experimental strategy is based on targeting NAc neurons that express c-Fos after CCI or CFA, but there is no comparison of the expression of c-Fos (or associated fluorescent proteins) between CCI/CFA and sham animals. Such a comparison would be welcome to assess the effect of CCI/CFA on NAc activity.

Minor
Abstract: The chronic pain models used should be mentioned in the abstract.

Results:
Please use the same scale on the Y-axis for similar experiments throughout the presentation of results. The lack of this standardization makes it difficult to compare data between different figures. Baseline values for PWT / L should be plotted in figures, as it was done in figure 4 B and C. Fig S1 E1-G1 there is no red plot or line Please indicate that FISH means (Fluorescent in situ Hybridization) in the first figure legend Methods: Why were the NAc injections unilateral? Is there any laterality between the injected side (NAc) and the side subjected to CCI/CFA (paw)? There is no mention to mice used in experiments of figure 5 (Cre) and 6 (black)  Please also see our responses to minor comment 4 from reviewer #2. Response 7: We thank reviewer for the kind suggestion. We perform control experiment to inject AAV8-Ef1a-DIO-eNpHR-mCherry only into POA and VTA respectively, without injection of pAAV1-PTRE-tight-NLS-Cre into NAc. We find no mCherry expression in POA and VTA in the absence of Cre ( Figure R1). The control condition is illustrated in new Supplementary Fig. S10a (Lazarus et al., 2011;Luo et al., 2018;Oishi et al., 2017;Tellez et al., 2012;Williams et al., 2020). Recently, Chen and his colleagues further classified the D1 and D2 MSNs into 30 D1 and 27 D2 subtypes by using single-cell RNA sequencing (Chen et al., 2021). They identified 7 subtypes neurons from NAc interneurons. These neuron subtypes may contribute to functional heterogeneity of the NAc. Besides sleepwakefulness and nociception regulation, the NAc also plays important roles in higher brain functions involving reward, sensitization, addiction, feeding, social, and depression-like behaviors (Francis et al., 2015;Kai et al., 2015;O'Connor et al., 2015;Ren et al., 2016;Smith et al., 2013;Tellez et al., 2012;Williams et al., 2020;Zhou et al., 2019). In our study, we found that the majority of CCIinduced NAc ensemble expressed dopamine D1 receptor (80.73 ± 1.72%), and the minority of CCI-induced NAc ensemble expressed dopamine D2 receptor (18.75 ± 1.20%). NAc non-ensemble neurons may be composed of D1R, D2R, oxytocin, and adenosine A receptors neurons. These neurons may be sleeppromoting, wake-promoting, or don't have correlation with sleep-wakefulness regulation. Consistently, the non-specific neurons in NAc from sham mice and unidentified NAc neurons exhibited a much higher degree of functional diversity, including many units that were more active during wake, NREM or REM sleep, respectively (Figure 1k-l). Some non-specific neurons were neither wake-active nor sleep-active neurons. Activating all of them may have opposite or no effects on sleep and wakefulness, and may not alter sleep-wakefulness behaviors.
In sham mice, we used the same tTA-TRE (Tet-Off)-based viral system in c-Fos-tTA mice as CCI mice (Figure 1a). And we terminated the doxycycline supply between day 0 and 21 after sham surgery, such that mCherry-tagged Channel Rhodopsin 2 (ChR2) were preferentially expressed in NAc neurons with high expression of c-Fos. The c-Fos expression level in NAc following sham surgery was significantly lower than that following CCI surgery ( Figure R10, please also see our responses to major comment 1b from reviewer #3). During 3 weeks after sham surgery, NAc neurons may be activated and tagged with ChR2 by feeding, social, locomotion, or sleep-wake behaviors (Luo et al., 2018;O'Connor et al., 2015;Oishi et al., 2017;Williams et al., 2020;Zhu et al., 2016).
Consistently, in our optrode recording, some tagged neurons in sham mice were neither wake-active nor sleep-active neurons (Figure 1k-l). Moreover, there was no significant difference in the mean firing rate of these tagged between wakefulness, NREM sleep, and REM sleep ( Figure 1k). These identified neurons may be related with feeding, social, or locomotion behaviors.
The mean firing rate of these neurons was not altered in sleep-wake transitions ( Figure S3a). Reactivating these neurons may not regulate sleep-wakefulness states at all, but modulate other behaviors. These have been discussed in the Discussion section on Page 18, line 5-22 and Page 19, line 1-2. Please also see our responses to minor comment 3.

Comment 2:
NAc is also involved in chronic pain regulation. A very recent study (Sato, 2022) indicates that optogenetic activation of NAc D1R neurons reversed the lowered pain thresholds due to neuropathic pain, the inconsistent findings with this study might be due to different animal models and manipulation of different populations of D1 neurons.
Nevertheless, NAc D1 neurons regulate chronic pain. The author should consider how to interpret their findings are different from previous studies, instead of saying 'Our results show that some of these sleep-regulating NAc neurons also regulate pain experience'. If the population of cells involved in the regulation of behavior also regulates the send relevant behavior, the significance of this study is discounted.

Response 2:
We thank the reviewer for pointing out this issue. Sato et al did laser stimulations at 30Hz of NAc D1R neurons to reverse the lowered pain thresholds. In our study, application of blue laser at 10Hz to NAc D1R neurons exacerbated pain-like (nociceptive) responses. The inconsistent findings may be due to different frequency stimulations. Previous study has shown that distinct frequency stimulations induced the frequency-dependent effects on cell firing in subthalamic nucleus and substantia nigra pars reticulata (Milosevic et al., 2018). Specifically, subthalamic firing attenuated with ⩾20 Hz stimulation (silenced at 100 Hz), while substantia nigra pars reticulata decreased with ⩾30 Hz (silenced at 50 Hz) (Milosevic et al., 2018)  The authors reported that only ~20% of D2 neurons are labeled by chronic pain, which is not well consistent with a previous study that chronic pain dramatically increased the AMPA/NMDA ratio in NAc D2 neurons (Ren, 2016), indicating an increase of D2 neuronal activity. Furthermore, 20% of neurons can play an important role in regulating both sleep and pain because activation of D2 neurons increases sleep (Oishi, 2017). Chronic pain activates D2 neurons and D2-neuron activation increases sleep are NOT in the context of chronic pain-induced sleep disorders. The author should carefully address this gap but not avoid talking about these issues.

Response 3:
We appreciate the reviewer's insight. Ren and his colleagues found the dramatically increase of the AMPA/NMDA ratio in NAc D2 neurons by ex vivo brain slices recording in SNI mice (Ren et al., 2016). In the same study, they also found the mEPSC frequency of NAc D2 neurons decreased in SNI mice (Ren et al., 2016). Beside, the increase of AMPA/NMDA ratio may not completely contribute to the increased firing and c-fos expressing in NAc D2 neurons in vivo.

Response 4a:
We appreciate the insightful issue. Luo et al did optogenetic activation of NAc D1 neuron terminals in VTA to increase wakefulness (Luo et al., 2018).
However, direct inhibiting VTA neurons that received the NAc ensemble inputs did not affect sleep and wakefulness in our study. These different results may be due to manipulating different neuronal populations via different neuronal pathways. Terminal activation has been proved to have adverse side effects (Zingg et al., 2017). Given that a brain region ''X'' is known to mediate a behavior/function of interest, determining which neural pathways downstream of X mediate this behavior/function remains challenging (Figure R4a-b).   Zingg et al., 2017).  in NAc and at axon terminals in VTA or LH induced wakefulness (Luo et al., 2018). Similarly, optogenetic activation of VTA Vglut2 neurons both at somas in VTA and at projections in NAc or LH promoted wakefulness (Yu et al., 2019).

Neurons in X project
Consistently, laser activation of CALCA (calcitonin gene-related peptide alpha) or CCK (cholecystokinin) neurons both at cell bodies in the pIII (perioculomotor) region and at axon terminals in POA or GiV increased NREM sleep (Zhang et al., 2019). Therefore, to avoid "antidromic spikes" and specifically inactivate NAc-ensembleinnervating VTA neurons, we applied anterograde trans-synaptic strategy to transsynaptic inhibit VTA neurons that received the NAc ensemble inputs in our study ( Figure R4 and Figure 7).   Figure R6a-b). Moreover, optogenetic stimulation (10 Hz x 2 min) of NAc D2 neurons increased the percentage of NREM sleep, and decreased wakefulness and REM sleep, resulting in an overall upregulation of NREM sleep ( Figure   R6c). Figure R6. Response 1: We appreciate the helpful suggestion from reviewer. To follow reviewer's request, similar to the experiment in the responses to major comment 3b, we performed optrode recording on D2 neurons specifically in NAc ensemble from c-fos-tTA-Tg mice ( Figure R7a-b). We combinated "Tet-Off" with "Cre-On" by injecting 2 AAVs (AAV-Tre3G-CRE-WPRE-pA and rAAV-D2-DIO-hChR2-mCherry-WPREs) into c-Fos-tTA mice two weeks before the CCI surgery.
In our study, about 80% NAc-ensemble are D1 neurons, the changes of their activity across sleep-wake transitions (Figure 1m) are consistent with previous study.   Figure 1F & 2D, a significant 10 Hz signal in EEG heatmap. Is it from optostimulation? However Figure 1L did not show it. Noise elimination is needed to increase the reliability of results.

Response 2:
We thank the reviewer for pointing out this issue. We think the 10 Hz signal in EEG heatmap in Figure 2f (not Figure

Figure R8. Optogenetic stimulations in different brain regions induce signal at stimulated frequency in EEG. a EEG power before (red trace) and after (blue trace) onset of photo-stimulation in NAc of D1-ChR2-mCherry (Left) or D1-mCherry (Right) mice.
20 Hz optogenetic activation of NAc D1 neurons induced a peak at 20Hz in EEG spectral power (modified from Luo et al., 2018).  Figure S3, the firing rate of NAc D1 neurons was not altered in sleep-wake transitions in naïve mice, do the author think these neurons are not involved in the regulation of wakefulness? How to understand the increased calcium activity of NAc neurons in wakefulness from the previous study?

Minor comment 3: In
Response 3: We appreciate the reviewer's insight. In Figure S3, we are not sure whether the labelled neurons in sham mice were D1 neurons. In sham mice, we used the same tTA-TRE (Tet-Off)-based viral system in c-Fos-tTA mice as CCI mice ( Figure 1a). During 3 weeks after sham surgery, NAc neurons may be activated and tagged with ChR2 by feeding, social, locomotion, or sleep-wake behaviors (Luo et al., 2018;O'Connor et al., 2015;Oishi et al., 2017;Williams et al., 2020;Zhu et al., 2016). Consistently, some identified neurons in sham mice were not typical sleep-or wake-active neurons (Figure 1k-l). Moreover, the mean firing rate of these identified neurons in sham mice was comparable during wakefulness, NREM, and REM sleep (Figure 1k). These identified neurons may be related with feeding, social, or locomotion behaviors. The mean firing rate of these neurons may be not altered in sleep-wake transitions. Please also see our responses to major comment 1. Figure 6H, why these VTA and POA-projecting cells are normalized to total

VTA and POA cells, but not NAc cells?
Response 4: We apologize if we did not make clear statement in the Figure 6 legend. In We also display the Figure R5 data as following: Figure R5.   (Fig 1).

The optogenetic stimulation of these neurons further decreases mechanical paw withdrawal threshold (PWT) and thermal latency (PWL); and
increases wakefulness (decreasing NREM sleep) in CCI or CFA animals (Fig 2 and 3).
Complementary, their optogenetic inactivation induces the opposite effects (Fig 4).  06). Finally, the authors used a dual-viral system to optogenetically inactivate the VTA or POA neurons innervated by the NAc targeted neurons.
Inactivation of POA neurons decreases sleep (with no effect on nociceptive tests), while inactivation of VTA neurons decreases PWT and PWL (with no effect on sleep) (fig 07). Based on this broad set of data, the authors concluded that these NAc-targeted neurons form "… a common NAc ensemble that encodes chronic pain and controls sleep".

The bidirectional relationship between negative changes in sleep and pain has been the focus of
several studies in the last two decades and the NAc is emerging as a key candidate to mediate the effect of decreased sleep on pain processing. However, the underlying mechanisms are poorly understood, and this is the first study to identify a neural population in the NAc that is apparently involved in the control of pain and sleep/wake cycle. These data will impact the literature on the field and are at the edge of knowledge. I have one main concern about the experiments and some suggestions to improve the manuscript reading and data presentation.

Response :
We appreciate the reviewer for your positive comments on our work.  (Figure. R9a). The EMG signals could be detected when the movement of paw withdrawal was induced by mechanical or thermal stimulation in CCI mice (Figure. R9b). Next, both optogenetic activation and inactivation were applied to NAc ensemble neurons in NAc-ensemble-ChR2

Major
and NAc-ensemble-eNpHR mice respectively, we did not find the significant change in EMG spectral power during blue or yellow laser stimulations ( Figure. R9c-9f) under free moving state. The reviewer kindly suggested to perform CPP task under target neurons inhibition. Since the mice had an optical fiber cable connected with laser, they could not move freely through the sliding door between two chambers in CPP task. We performed open field test so that the mice with optical fiber cables could move freely in a large chamber. The total distance traveled by NAc-ensemble-ChR2 (or NAc-ensemble-eNpHR) mice was compared during laser off and on period. Both blue and yellow laser stimulations have no effect on the total distance traveled by NAc-ensemble-ChR2 and NAc-ensemble-eNpHR mice, respectively ( Figure R9g-9h).

REVIEWER COMMENTS</B>
Reviewer #1 (Remarks to the Author): The authors are highly responsive to reviewers' critiques. They have performed extensive additional experiments to validate their conclusions as well as to tease apart the important differences between their findings and previously published results from various other groups. These additional results will be highly informative to the neuroscience community for guiding the proper usage of optogenetics, as well as to better understand the NAc functional neuronal ensembles. They should be all incorporated into the supplementary (or even main) figures rather than only used to show the reviewers. Related results and discussions should be also incorporated into the main text. Detailed suggestions are listed below.
Response #1: This is an important clarification that needs to be incorporated into the main text. A single citation without explanation is not sufficient, as readers outside of the pain field would not immediately understand the c-Fos expression scenario here.
Response #6: Fig. 6H: mathematically, if POA+/total ensemble is ~ 80%, and VTA+/total ensemble is ~ 80%, then the overlapping subpopulation, e.g. POA+VTA+/total ensemble, should be at least 60% (80%+80%-100%=60%). The fact that it was much lower (~30%) is puzzling and needs to be addressed.  Reviewer #2 (Remarks to the Author): In the revised manuscript, the authors have well addressed my comments with new experiments, discussion, and illustration of previous data. I do believe that this vision has substantial improvements and seems close to be published. A few minor comments to the authors for their consideration are listed below. 1. In Comments 1, the authors discussed the heterogeneity of NAc non-ensemble neurons to explain why activation of these non-ensemble did not alter sleep-wake behaviors. However, it is better to tune down this conclusion because activation of PVT-or VTA-projections in the NAc are also wake-promoting (these are all non-specific activation). In general, the authors met my requests.
The experiment of c-Fos expression in NAc three weeks after sham or CCI surgery is adequate (Figure R 10). I believe neither the EMG data ( Figure R9 c and d) nor the open field data ( Figure R9 g and h) are golden pattern experiments to meet my concern, but they are acceptable. The justification for not including a nociceptive test without motor bias is invalid, first because the CPP could be done with chemogenetics and second because this was just a suggestion, there are other test options. But this reviewer understands the resistance to performing experiments with totally new methodologies in an already experimentally robust article. What this reviewer do not understand is why the authors did not include the experiments performed to follow the reviewers` requests on the MS or supplementary material. It seems that the authors simply dismissed all the queries and suggestions of the reviewers as preliminary or useless (except for Fig R1  which was included). I'm not suggesting including all the experiments, evidently some of them are not strictly related to MS, but I would like to see at least the controls I requested and the experiments with D2 neurons (but note that Figure R 3 is not shown with fig R 6 being repeated in its place).

Reviewer #1 (Remarks to the Author):
The authors are highly responsive to reviewers' critiques. Response 1: We thank the reviewer for the kind suggestion. We add "The c-Fos has been used as a neural marker of pain over several decades since Hunt et al . CCI mice exhibited persistent pain hypersensitivity at least for 3 weeks. The c-Fos expression level in NAc three weeks after CCI surgery was dramatically higher than that after sham surgery (Supplementary Fig. 2a-b).
Recently, the tTA-TRE (Tet-Off)-based viral system has been applied to label itch-and pain-specific neurons (Jiang et al., 2022)." in the results on Page 5, line 8-13.

Response 2:
We appreciate the reviewer for the kind suggestion. We change "alertness" for "arousal" in the discussion on Page 23, line 16.

Response 4:
We thank the reviewer for the good suggestion. Figure R2 is incorporated into supplementary materials as the new Supplementary Fig. 11. The duration of individual laser pulse is 5 ms, that is described in the supplementary legends on Page 15, the last two lines. The result is described in main text on Page 13, line17-21, and Page 14, line 1-4.

Reviewer #2 (Remarks to the Author):
In also wake-promoting (these are all non-specific activation).

Response 1:
We thank the reviewer for the kind suggestion. To follow the reviewer's suggestion, we tune down the conclusion. We delete the sentence "instead of sleep and pain" in the discussion on Page 21, line 21. We add "On the other hand, the number of NAc non-ensembles neurons was relatively less compared with NAc ensemble neurons ( Fig. 2b, 2h). It is also possible that activation of non-ensembles neurons (a small subset of neurons) might not affect sleep-wakefulness or pain response." in the discussion on Page 22, line 2-5.

Comment 2: Comments 2, if the different frequencies of stimulation induced the controversial results,
it should be mentioned in the current study.

Response 2:
We thank the reviewer for the good suggestion. Figure R2  We apologized that we made error in Figure R3 in the last rebuttal letter. We showed Figure   R6 twice but not Figure R3. The following is Figure R3. Figure R3 is incorporated into new Fig. 6.

Reviewer #3 (Remarks to the Author):
In general, the authors met my requests.

Comment:
The experiment of c-Fos expression in NAc three weeks after sham or CCI surgery is adequate (Figure R 10). I believe neither the EMG data ( Figure R9 c and d) nor the open field data ( Figure R9 g and h) are golden pattern experiments to meet my concern, but they are acceptable.  R 6 being repeated in its place).

Response:
We thank the reviewer for the good suggestion. Figure R10 (The c-Fos expression in NAc three weeks after sham or CCI surgery) is added in the new Supplementary Fig. 2. This is described in the results on Page 5, line 10-12.
Please also see our response to comment 1 from reviewer #1. Figure R9 ( Please also see our responses to comment 5 from reviewer #1 and comment 3 from reviewer #2.
We apologized that we made error in Figure R3 in the last rebuttal letter. We showed Figure   R6 twice but not Figure R3. The following is Figure R3. Figure R3 is incorporated into new Fig. 6.